SURFACE BASED PROBES OF IRON/FERRITIN INTERACTIONS

铁/铁蛋白相互作用的表面探针

• 批准号: 2749524
• 负责人: MICHAEL J. NATAN
• 金额: $ 12.73万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2749524
• 关键词: Raman spectrometry; active sites; chemical binding; ferritin; ferroxidase; high performance liquid chromatography; intermolecular interaction; iron; molecular site; protein structure function; site directed mutagenesis; temperature; tyrosine

DESCRIPTION: Changes in iron homeostasis have dramatic consequences for human health. Cancer, heart disease, neural disorders - even aging itself - have been linked to excess iron or iron-induced oxidative damage. Storage of ferrous (Fe(2+) iron in a non-toxic (Fe(3+) mineral oxide form (FeOOH) by ferritin (Ft), a spherical, hollow protein, thus assumes tremendous significance in human physiology. Organ-specific modulation of the rate of iron uptake by Ft, a long-term goal with enormously favorable biomedical implications, presupposes a basic understanding of the uptake mechanism. Unfortunately, several aspects of iron traversal across the 20-A thick protein sheath are not completely understood. These include the iron coordination environment during oxidation, transport, and mineralization, as well as mechanistic differences between protein L and H subunits. Detailed information about the iron coordination sphere at (and within) the protein surface over the course of uptake and biomineralization will be generated using surface enhanced Raman scattering (SERS), a vibrational spectroscopy with extraordinary sensitivity for molecules in proximity to roughened Ag surfaces. The use of Raman spectroscopy to provide detailed molecular-level information about non-heme iron binding sites in proteins is extremely well-documented, as are numerous studies demonstrating retention of Ft quaternary structure when adsorbed to metal surfaces. Accordingly, preliminary SERS studies at two different excitation wavelengths of well-defined Fe:Ft complexes (0.5, 2,4, and 6 Fe: subunit) adsorbed to SERS-active colloidal Ag surfaces reveal dramatic spectral differences that clearly emanate from distinct iron-containing species. Detailed information of this nature on stable intermediates in the biomineralization of iron by ferritin has never before been available at physiological temperatures. The proposed work will expand on these exciting results to generate a step-by-step molecular view of the early stages of iron uptake by Ft, with a particular focus on elucidating the amino acids involved in oxidation and transport. Experiments on well-defined Ft samples from several organisms, with varying H/L ratios, different ligand sets (i.e. site-directed mutants), and different Fe:Ft ratios will be studied as a function of excitation wavelength, temperature, and iron oxidant (inner sphere vs. outer sphere). This research will not only greatly improve understanding of iron homeostasis but demonstrate the utility of SERS as a powerful probe of metalloprotein structure and function.

描述：铁稳态的变化对 人类健康。 癌症，心脏病，神经疾病 - 甚至自我衰老 - 与铁或铁诱导的氧化损伤有关。 贮存 通过非毒性（Fe（3+）矿物氧化物形式（FeOOH）的铁（Fe（2+）铁） 铁蛋白（ft），一种球形的空心蛋白，因此假定很大 人类生理学的意义。 器官特定的调制速率 FT的铁吸收，这是一个长期目标，具有极大的生物医学 含义，以对吸收机制的基本理解为前提。 不幸的是，跨20-A厚的铁遍历的几个方面 蛋白质鞘尚未完全了解。 这些包括铁 氧化，运输和矿化过程中的协调环境AS 以及蛋白L和H亚基之间的机械差异。 有关铁协调领域（和内部）的详细信息 在吸收过程中蛋白质表面和生物矿化过程将是 使用表面增强的拉曼散射（SERS）生成的振动 光谱学对分子的敏感性非常敏感 粗糙的Ag表面。 拉曼光谱法提供详细的 有关蛋白质中非血红素铁结合位点的分子级信息是 非常有据可查的人，许多研究证明了保留率 当吸附到金属表面上时，ft第四纪结构的结构。 因此， 在两个不同的激发波长的初步研究 定义明确的Fe：FT复合物（0.5、2,4和6 Fe：Subunit）吸附到 SERS活性胶体Ag表面揭示了巨大的光谱差异 显然是由不同的含铁物种散发出来的。 详情 这种性质在稳定中间体的生物矿化中 在生理温度下，铁蛋白从未有过。 拟议的工作将扩大这些令人兴奋的结果，以产生 ft的铁吸收早期摄取的早期阶段的分子分子视图， 特别着重于阐明涉及氧化的氨基酸和 运输。 从几个生物体定义明确的FT样品进行的实验， 随着H/L比的不同，配体套件（即定点突变体）， 将根据激发的函数研究不同的Fe：ft比率 波长，温度和铁氧化剂（内球与外球体）。 这项研究不仅会大大提高对铁的理解 体内平衡，但证明了SERS的实用性作为强大的调查 金属蛋白结构和功能。